Skeletal muscle insulin resistance underlies the metabolic syndrome and the development of type 2 diabetes (T2D). More than 26 million US adults have T2D. Defects in phosphorylation events in insulin signaling are considered to be among the main causes of insulin resistance, however, the mechanisms for these defects remain elusive, which severely limits effective strategies to prevent and treat T2D. Most research on phosphorylation events in insulin resistance has been focused on kinases. Serine/threonine protein phosphatase 1 regulatory subunit 12 (PPP1R12) modulates the activity and specificity of the catalytic subunit of protein phosphatase 1 (PP1c), and anchors PP1c to its substrates. Nonetheless, little is known about the dephosphorylation events and functions controlled by PPP1R12 in the context of skeletal muscle insulin resistance. We hypothesize that protein abundance of PPP1R12 is lower and protein-protein interactions involving PPP1R12 are abnormal in skeletal muscle of obese non-diabetic and/or T2D subjects compared to lean controls, and that the lower PPP1R12 abundance contributes to the development of insulin resistance and T2D. Our hypothesis is based on the existing literature, and on our novel preliminary findings revealing that: 1). PPP1R12 protein abundance was lower in T2D skeletal muscle (6-fold vs. lean, P<0.05); 2). Multiple PPP1R12 interaction partners in skeletal muscle had altered associations with PPP1R12 in obese and/or T2D subjects vs. lean controls; 3). PPP1R12 knockdown in L6 cells led to increased phosphorylation for more than 100 sites on multiple proteins; 4). PPP1R12 knockdown resulted in reduced glucose uptake. To test our hypotheses, we propose to: 1. Determine PPP1R12 protein abundance and protein interaction partners in skeletal muscle from lean healthy controls, obese non-diabetic controls, and obese T2D subjects. We will perform a hyperinsulinemic-euglycemic clamp to assess insulin sensitivity, and obtain two skeletal muscle biopsy samples, one before the clamp and the other at the end of the clamp. PPP1R12 abundance and interaction partners will be quantified in muscle samples using proteomics and western blotting. 2. Determine how knockdown or overexpression of PPP1R12 affects insulin action in primary skeletal muscle cells from lean healthy controls, obese non-diabetic controls, and obese T2D subjects. We will culture primary skeletal muscle cells from biopsies from these subjects, and knockdown or overexpress PPP1R12 in these cells. Phosphoproteomics and classic insulin action measurements will be used to identify novel phosphorylation and signaling events regulated by PPP1R12. Our overall goal is to address the knowledge gap of phosphatase role in insulin resistance and T2D, using a combination of clinical studies (directly measure human pathophysiology), in vitro cell studies (for causal mechanisms), and cutting-edge proteomics (for global analysis of cell signaling & unbiased discovery). The outcome of this research will provide novel insights into the pathogenesis of insulin resistance, facilitating the design of better drugs for T2D prevention and treatment.